1. Field of the Invention
The present invention relates to a method of detecting injured cell nuclear DNA. More in detail, it is directed to the method for the detection of qualitative change of the cell nuclear DNA induced by carcinogenesis or like, and may be utilized in screening of cancer or malignant cells and for pathological diagnosis of cancer, or in a test for confirming the presence or absence of chemical carcinogenic substance.
2. Description of Prior Art;
It has been well known in the art that the cell nuclear DNA is quantitatively changed by aging or carcinogenesis, and the method for the detection thereof has already been established. However, it has been known that the cell nuclear DNA suffers the following qualitative alterations before it suffers the clear change as aforementioned. For example, known DNA alterations include:
1) Minute distortion due to alkylation of DNA base; PA1 2) Small distortion due to hydration or break off of DNA base; PA1 3) Large distortion induced by the interaction of a chemical substance (acting as an adduct) which has a large molecular weight to be interposed inbetween the DNA base linkage while forming a covalent bond; PA1 4) Breakdown at the straight chain portion of DNA induced by the formation of a double-stranded DNA dimer, which is formed by coupling of two bases, or the formation of a cross-linking bond between the double-stranded DNAs or a cross-linking bond between the DNA chain and a protein; and PA1 5) Injury of the nuclear DNA, e.g. shearing of double strands.
Development of a method of detecting such injured cell nuclear DNA is earnestly demanded, as a natural demand, and various methods have been proposed. For example, there is known a method in which the auto-repair of injured DNA, which is the characteristic for repairing injured DNA in the cell nucleus, is utilized so that nucleotide labelled with a radioactive isotope is incorporated in the repaired DNA followed by detection of the nucleotide labelled with the radioactive isotope. However, this known method has some problems that a special equipment is needed due to the use of radioactive isotopes, and that the capacity of the equipment is limited.
The method precedingly proposed by the inventor in Unexamined Japanese Patent Publication No. 8053/1987 is a method in which a single-stranded DNA is prepared from a cell suspension sample or from a fixed tissue specimen on a glass slide by proper method, and then the stainability by the use of acridine orange is examined. This method is based on the finding that injured nuclear DNA is selectively hydrolyzed by treating the same with an acid under an appropriate condition to produce single-stranded DNA. Based on this finding, the proposed method utilizes differential fluorescent staining of cancerous cell by using acridine orange. When acridine orange binds to double-stranded DNA as a manner of intercalation, it emits orthochromatic green fluorescence. When acridine orange binds to single-stranded DNA by stacking, it emits red-shifted metachromatic fluorescence due to dye aggregation.
In general, the tissue samples prepared for the pathological diagnosis are samples or specimens fixed with formalin. DNA strand might be injured occasionally leading to trivial injury thereof during the step of fixation with formalin to form DNA strands which are resulted by partial unlinking. In the method proposed by Unexamined Japanese Patent Publication No. 8053/1987, acridine orange may stack in to the single-stranded portions of DNA which have been formed partially with such trivial distortion. As a result, there arises another problem that the specimen fixed with formalin prepared for the pathological diagnosis suffers "pseudo-positive" stainability and thus a smear specimen prepared by grinding a fresh tissue must be used as the sample to be inspected.
It is preferable to digest the sample with RNase prior to acid-hydrolysis of DNA to remove RNA. By the addition of this digestion step, it becomes possible to prevent occurrence of "pseudo-positive" stainability resulted from the conjugation of RNA with the anti-single-stranded DNA antibody.